EP0556214A1 - Procede d'amplification de l'acide nucleique - Google Patents

Procede d'amplification de l'acide nucleique

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Publication number
EP0556214A1
EP0556214A1 EP91918606A EP91918606A EP0556214A1 EP 0556214 A1 EP0556214 A1 EP 0556214A1 EP 91918606 A EP91918606 A EP 91918606A EP 91918606 A EP91918606 A EP 91918606A EP 0556214 A1 EP0556214 A1 EP 0556214A1
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EP
European Patent Office
Prior art keywords
hla
primer
sequence
specific
nucleic acid
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EP91918606A
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German (de)
English (en)
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EP0556214B1 (fr
Inventor
Adrian Vivian Sinton Hill
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Chancellor Masters & Scholars Of University
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University of Oxford
British Bio Technology Ltd
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Priority to EP98108382A priority Critical patent/EP0875582B1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a process for amplifying a .desired, nucleic acid sequence, and in particular for the amplification of a nucleic acid sequence encoding at least part of a human leucocyte antigen.
  • HLA Human leucocyte antigen
  • PCR polymerase chain reaction
  • PCR techniques themselves are known from EP-A-0200362, EP-A-0201184 and EP-A-0258017, all of which are in the name of Cetus Corporation. These publications disclose processes for amplifying a desired nucleic acid sequence by treating separate complementary strands of nucleic acid with a molar excess of two oligonucleotide primers, and extending the primers (often using Klenow fragment) to form complementary primer extension products which act as templates for synthesising the desired nucleic acid sequence.
  • EP-A-0258017 teaches that the use of DMSO in an amplification buffer is undesirable when using a heat stable polymerase such as Tag polymerase as it- inhibits the activity of the polymerase, and that the addition of EDTA will halt amplification by inactivating the polymerase.
  • a heat stable polymerase such as Tag polymerase
  • the primer may loop back on itself (to form a hairpin) or form a complex structure, both of which will prevent amplification of the desired sequence.
  • the primer may hybridise to a different sequence from the one that is desired to amplify. (This problem is also encountered when designing probes for a- specific sequence.)
  • HLA is the human version of the major histocompatibility complex (MHC) which is a cluster of genes that encode for cell surface antigens on leucocytes. These antigens are responsible for rejection of skin and organ grafts between individuals.
  • MHC major histocompatibility complex
  • class I (covering types A, B and C) ;
  • the present invention seeks to overcome or at least mitigate some or all of the above problems, and provide a PCR technique that may allow the typing and sub- typing of human leucocyte antigens.
  • a process for amplifying a nucleic acid sequence comprising two complementary strands (which may or may not be hybridised) , at least one strand coding for a human leucocyte antigen (HLA) sequence, the process comprising:
  • each extension product when it is separated from its complement, can serve as a template for synthesis of an extension product of the other primer, at least one of the extension products comprising the HLA sequence;
  • a polar aprotic solvent such as dimethylsulphoxide (DMSO)
  • HLA sequences and alleles may be grouped into types and sub-types by being able to successfully amplify HLA sequences.
  • the Applicants believe that the very high GC nucleotide content of HLA sequences, especially class I sequences which can be from 70 to 75% GC rich, may be responsible for previously unsuccessful attempts to employ PCR in HLA typing. It is believed that the use of a buffer containing a polar aprotic solvent such as DMSO during amplification allows amplification of such (often GC rich) sequences to be viably repeated.
  • two primers are employed (one specific for each of the two strands of the nucleic acid sequence to be amplified) .
  • the HLA nucleic acid sequence suitably comprises a sequence from the class I, II or III alleles, although class T is the class of choice.
  • the present invention has been found to be particularly useful for amplifying class I sequences as they are often GC rich.
  • Suitable class I sequences include sequences that belong to type A, B or C, and preferably type B.
  • stages (b) to (d) are repeated at least once. Repetition for at least 20, and preferably 30, times is generally preferred.
  • the stages, (b) and (c) are suitably accomplished by treatment with the four different nucleotide triphosphates and polymerisation agent, such as DNA polymerase I, a Klenow fragment of DNA polymerase I, T4 DNA polymerase or reverse transcriptase.
  • the polymerisation agent of choice is a heat-stable enzyme such as a Ta ⁇ polymerase (available from Cetus Corporation, 1400 53rd Street, Emeryville, California 94608, U.S.A.). Efficient amplification has been achieved with this enzyme despite the fact that EP-A-0258017 teaches that DMSO inhibits its polymerase activity.
  • the oligonucleotide primers are preferably specific for at least a portion of an alpha domain of the HLA gene, such as alpha 2 or (preferably) alpha 1, suitably found in the HLA B-type.
  • the primers are preferably less than 30, and more particularly less than 20, nucleotide in length.
  • Suitable primers are those that are specific for at least a portion of the nucleic acid corresponding to any or all of the following regions:
  • residues 1 to 50 such as 1 to 30, and optimally 1 to 10, of the alpha 1 domain;
  • residues 51 to 93 such as 60 to 90, of the alpha 1 domain
  • residues 101 to 150 such as 105 to 130, optimally 110 to 125, of the alpha 2 domain.
  • the phrase "corresponding to” means, unless the context requires otherwise, that the nucleic acid codes for, or is complementary to nucleic acid coding for, the amino acids or residues specified.
  • primers are capable of hybridising to (and will thus be complementary, or substantially complementary, to) a nucleic acid sequence corresponding to (in the sense of coding for, or being complementary to, nucleic acid coding for) any or all of the following regions:
  • residues 2-8 and/or residues 85-90 which primers can be used to amplify HLA class I alleles, types A, B and C;
  • residues 2-8 and/or residues 67-72 which primers can be used to amplify HLA-B27 alleles
  • residues 61-67 and/or residues 114-120 (which primers can be used to amplify HLA-BW53 and HLA-B35 alleles); which are all present in the alpha 1 or alpha 2 domain of HLA.
  • Preferred examples of the above primers have the sequences:
  • Particularly preferred primers are capable of selectively hybridising to (and thus amplifying) a sequence:
  • HLA class I gene such as type A, B or C, and preferably type B
  • HLA-Bw53 and HLA-B35 gene or single alleles e.g. HLA-B*2703 .
  • Suitable primers include those that are capable of hybridising to a nucleic acid sequence, or its complementary sequence, at least one of which encodes any or all of the following amino acid sequences:
  • HSMRYF SEQ.ID:6
  • YNQSEA SEQ.ID:7
  • HSMRYF SEQ.ID:6
  • CKAKAQ SEQ.ID:8
  • the primers used in the present application are thus suitably specific for (and thus the HLA sequence to be amplified is present in) :
  • alleles only in a specific group i.e. allele-group-specific (e.g. HLA-B27 group or the group containing HLA-Bw53 and HLA-B35) ; or
  • all alleles or only a chosen type, group or allele can be amplified.
  • the present invention allows the typing of HLA alleles.
  • the primers employed are either allele-group-specific or allele-specific. Particularly but not exclusively in the latter case, detection of and/or identification of an allele may be used in the diagnosis of disease or predisposition to disease.
  • the buffer suitably contains from 1% to 20%, preferably from 5 to 15% and optimally from 8 to 12%, of the polar aprotic solvent.
  • solvents include acetonitrile, DMF and acetone although the solvent of choice is dimethylsulphoxide (DMSO) .
  • the buffer also suitably contains an amount of each of the four deoxynucleotide triphosphates (dATP, dCTP, dGTP and dTTP) such as at a concentration of from 100 to 300 mM, preferably from 150 to 250 mM, and optimally from 180 to 220 mM.
  • the primers are suitably provided in an amount of from 30 to 80, preferably from 40 to 60, pmol.
  • the buffer is suitably at a pH of from 6 to 10, optimally from 8.5 to 9.0. The same buffer is suitably employed for all stages (a) through to (d) .
  • the buffer preferably contains magnesium ions, for example supplied as magnesium chloride MgCl 2 , such as in an amount of from 0.5 to 5 mM, preferably from 1 to 4 mM.
  • the buffer advantageously contains ammonium ions, such as provided by ammonium sulphate, which has surprisingly been found to assist amplification.
  • the ammonium salt is suitably provided in an amount of from 10 to 50 mM, such as 15 to 30 mM, and optimally from 16 to 18 mM.
  • the buffer will be provided with a chelating agent.
  • the chelating agent assists amplification somewhat unexpectedly due to the fact that it may complex MgCl 2 which is often employed in the buffer.
  • a chelating agent such as EDTA inactivates the polymerase.
  • the chelating agent may be present in an amount of from 1 to 110 mM, preferably from 20 to 80 mM, and optimally from 60 to 70 mM.
  • Preferred chelating agents include polyacetic acids, for example polyamine polyacetic acids such as ethylenediaminetetraacetic acid (EDTA) .
  • the nucleic acid used in the process of the present application is suitably DNA, and preferably genomic DNA.
  • the nucleic acid will generally be double-stranded. If it is, then it is preferred that the strands are separated by denaturation before or during stage (a) .
  • the double stranded nucleic acid can be suitably denatured by heating at a temperature of from 90-110°C, such as at from 1 to 10 minutes.
  • the same conditions are preferably employed in stage (c) .
  • stages (a) through to (d) may be performed simply by varying the temperature of the buffer, such as by using a thermal cycling apparatus.
  • the temperature of the buffer can be particularly important. If the temperature is too low, the primers may anneal to many different -sequences in the nucleic acid, resulting in non-specific amplification. Too high a temperature may result in insufficient hybridisation and thus no amplification.
  • Stage (b) is preferably conducted at a temperature of from 41 to 70°C, preferably from 45 to 63°C, and optimally at from 50 to 58°C.
  • the exact temperature employed may depend upon the primers being employed and other operating conditions. Thus, for the preferred primers whose specific sequences have been mentioned, the corresponding preferred temperature employed in stage (b) are as follows:
  • the temperature is suitably from 50 to 80°C, such as from 30 seconds to 2 minutes.
  • the invention finds particular use in the detection of, or detection of predisposition of, diseases or disorders including inflammatory arthropathy such as arthritis, and arthritis related diseases, including reactive arthritis, Reiter's syndrome, uveitis and in particular ankylosing spondylitis.
  • diseases or disorders including inflammatory arthropathy such as arthritis, and arthritis related diseases, including reactive arthritis, Reiter's syndrome, uveitis and in particular ankylosing spondylitis.
  • the process of the first aspect of the invention preferably additionally comprises:
  • the label is preferably a radioactive one, such as one containing 32 P. This may be attached to the probe by using gamma 32 P dATP (with T4 polynucleotide kinase) .
  • Preferred probes are from 10 to 30 nucleotides in length, such as from 15 to 20 nucleotides.
  • the probe is preferably specific for at least a portion of the alpha 1 domain of the HLA sequence, such as HLA type B.
  • the probe is a primer as mentioned before, and so the preferred features and characteristics of the primers are as for the probe mutatis mutandis.
  • Suitable probes are those that are specific for at least a portion of the nucleic acid corresponding to any of the following regions:
  • residues 70 to 90 such as 75 to 88, of the alpha 1 domain
  • residues 50 to 80 such as 55 to 64 and/or 65 to 74 of the alpha 1 domain.
  • Particularly preferred probes have nucleic acid a sequence that comprises a sequence that is specific for a sequence that encodes CX 1 X 2 KA wherein each of X 1 and X 2 individually represent any amino acid, or the complementary nucleic acid sequence.
  • Such probes are suitably capable of hybridising to nucleic acid corresponding to the 67-71, e.g. 66-72 region of the alpha 1 domain (HLA-B27 and thus specific only for. the group) .
  • X 1 represents K and/or X 2 represents A.
  • a suitable probe is 5'- CTGCAAGGCCAAGGCACA-3' (SEQ.ID:11) .
  • probes have a sequence that is capable of hybridising to a sequence spanning the sequence encoding residue 59 (H:His) in the alpha 1 domain (of HLA-B) , e.g. residues 58 to 60, 57 to 61 or 56 to 62, such as one that encodes the polypeptide sequence GPEHWD (SEQ.ID:14).
  • Such probes are suitable for identifying HLA-B*2703.
  • a suitable probe is 5'- GGGCCGGAGCATTGGGAC-3' (SEQ.ID:12) .
  • a further suitable probe has a sequence that comprises a sequence that is specific for a sequence that encodes a Bw4 epitope, e.g. IALR, or the complementary nucleic acid sequence.
  • a suitable probe is 5 ' - CGGATCGCGCTCCGCTAC-3' (SEQ.ID:13) .
  • the present invention thus encompasses a method of detecting and/or identifying an HLA sequence that may be indicative of a patient's susceptability to inflammatory arthropathy, such as ankylosing spondylitis, the method comprising amplifying nucleic acid by using the process of the first aspect of the present invention where the nucleic acid comprises an HLA sequence, such as a sequence from the alpha l domain, hybridising to the HLA sequence a detectably labelled probe, and detecting any hybridised nucleic acid so formed.
  • the probe may be radio-labelled, metal labelled, or enzymatically labelled.
  • the nucleic acid comprising the HLA sequence will generally be present in a sample, which sample is preferably obtained from DNA extracted from white blood cells.
  • the detection of hybridisation may thus indicate either the detection and/or identification of an HLA sequence and/or a specific type or sub-type of HLA sequence.
  • the primer and/or probe may be specific for only one allele, for example HLA-B*2703. The detection and identification of this allele may indicate that a human is not susceptible to arthritis.
  • the present invention can therefore be thought of in another aspect as relating to a process for amplifying a human leucocyte antigen nucleic acid sequence, the sequence being contained in nucleic acid consisting of two complementary strands, which may be of equal or unequal length, the process comprising:
  • Stages (a) to (c) are repeated at least once, and preferably from 20 to 40, such as from 30 to 35, times.
  • a second aspect of the present invention relates to a kit for the detection and/or identification of a human leucocyte antigen (HLA) sequence (for example that may be indicative of the risk of a human being susceptible to inflammatory arthropathy, e.g. arthritis or an arthritis related disease) in a sample, the kit comprising:
  • the primer and/or the probe is specific for an allele group only (e.g. HLA-B27) or (preferably) specific for only one allele (e.g. HLA-B*2703) .
  • the kit may thus be used to determine the risk, such as a high or low risk, of a human to a disease or predisposition to disease.
  • the kit can be used for determining whether a human has a high or low risk to inflammatory arthropathy, e.g. arthritis or an arthritis related disease (low risk may be indicated by the presence of the HLA-B*2703 allele) .
  • a third aspect of the present invention relates to a nucleic acid sequence that is a primer or a probe as discussed for the first aspect.
  • a fourth aspect of the present invention relates to a method of detecting if a human has, or .has predisposition to, a disease, for example being susceptible to a high or low risk inflammatory arthropathy, e.g. arthritis, such as ankylosing spondylitis , the method comprising conducting a process according to the first aspect and determining the presence of a HLA sequence indicative of a disease, or predisposition to a disease , such as inflammatory arthropathy.
  • a disease for example being susceptible to a high or low risk inflammatory arthropathy, e.g. arthritis, such as ankylosing spondylitis
  • inflammatory arthropathy includes rheumatic disease, arthritis such as osteoarthritis , rheumatoid arthritis , reactive arthritis , Reiter' s syndrome , uveitis , ankylosing spondylitis , viral arthritis , psoriatic arthropathy , gouty arthritis , septic arthritis (suppurative arthritis) , erythema nodosum and Henoch-Schloelein purpura.
  • arthritis such as osteoarthritis , rheumatoid arthritis , reactive arthritis , Reiter' s syndrome , uveitis , ankylosing spondylitis , viral arthritis , psoriatic arthropathy , gouty arthritis , septic arthritis (suppurative arthritis) , erythema nodosum and Henoch-Schloelein purpura.
  • Ankylosing spondylitis is often characterised by the gradual onset of low back pain (sometimes bilateral buttock pain) with morning stiffness . Peripheral j oints may be affected . Often a reduced range of spinal movement and chest expansion is experienced, followed by rigidity of the spine, often in the cranial direction (firstly lumber, then dorsal , then cervical) . Clinically one may often find high dorsal kyphosis , obliteration of lumber lordosis and flattening of the chest.
  • the method of the fourth aspect suitably involves is ol at ing DNA from wh ite bl ood cel l s b e f ore amplification according to the first aspect .
  • the method also preferably involves the stages (e) and (f) of the first aspect. This is suitably conducted by dot blot analysis .
  • a fifth aspect of the present invention relates to a process for the preparation of a nucleic acid sequence of the third aspect, the process comprising coupling successive nucleotides together and/or ligating successive oligo- and/or polynucleotides together.
  • the nucleic acid is preferably made by chemical synthesis. For example, this may be achieved employing diethylphosphoramidites, and preferably using automated synthesis.
  • the procedure suitably involves detritylation with trichloroacetic acid in dichloromethane, condensation with benzotriazole, and capping with acetic anhydride and dimethylamino pyridine in tetrahydrofuran and pyridine.
  • FIGURE 1 shows the amino acid sequences of residues 1 to 90 of the alpha 1 domain of a number of HLA-B alleles
  • FIGURE 2 shows the amino acid sequences of residues l to 273 of the alpha 1 domain of a number of HLA-B alleles
  • FIGURE 3 shows the amino acid sequences of residues 50 to 120 of the alpha 1 domain of a number of HLA-B alleles and two primers, and one probe, of the present invention
  • FIGURE 4 shows the amino acid sequences of residues l to 90 of the alpha 1 domain of several HLA-B27 alleles. probes of the present invention that are specific for HLA-B*2703 and HLA-B27 group alleles and two primers of the present invention for amplification of HLA-B27 group alleles; • and
  • FIGURE 5 shows the results of dot blot analysis using the HLA-B27 detection probe.
  • a process for amplifying a nucleic acid sequence (which may or may not be hybridised) ,at least one strand coding for a human leucocyte antigen (HLA) sequence, the process comprising:
  • the chelating agent is preferably a polyacetic acid, such as a polyamine polyacetic acid. Particularly preferred is EDTA.
  • the buffer preferably contains magnesium ions which are suitably provided by means of a magnesium salt, for example magnesium chloride.
  • a magnesium salt for example magnesium chloride.
  • the source of ammonium ions may be provided by way of an ammonium salt, such as ammonium sulphate.
  • a seventh aspect of the present invention relates to a composition suitable for use as a buffer in a polymerase chain reaction amplification process, the composition comprising:
  • HLA-B*2703 The polymerase chain reaction and oligonucleotide hybridisation was used to demonstrate that the predominant sub-type of HLA-B27 in the Gambia, West Africa is HLA-B*2703 which has never been found in other ethnic groups. This sub-type differs from the common Caucasian HLA-B27 sub-types in its recognition by cytotoxic T cells. Unlike other sub-types HLA-B*2703 has not been shown to be associated with ankylosing spondylitis and the predominance of this sub-type may in part account of the rarity of this condition in black populations.
  • a panel of 180 well-defined antisera were used to type either freshly isolated or cryopreserved peripheral blood lymphocytes (PBL) using the standard 2-stage NIH microcytotoxicity assay. Following removal of peripheral blood lymphocytes, DNA was extracted from the residual white cells by lysis in a non-ionic detergent, overnight incubation in proteinase K -and two* phenol-chloroform extractions followed by one chloroform extraction. The DNA was precipitated in ethanol, redissolved in water and its concentration measured by spectrophotometric absorbance at 260 nm. For HLA-B27 typing oligonucleotide primers to conserve sequences at either end of the exon encoding the alpha 1 domain of HLA class I sequences were used (see Figure 4) . The primers used were:
  • the primers were synthesised using the procedure of Beaucage and Caruthers, Tetrahedron Letters 24: 245 (1981) . Amplification was performed in a buffer containing 3 mM MgCl 2 , 17 mM ammonium sulphate, 66 mM Tris of pH 8.8, 67 mM EDTA, 10% DMSO, ImM spermidine, 200 microM of each dNTP with 50 pmol of each primer and 0.5 meg of DNA in a 50 microlitre reaction volume.
  • Example 1 The procedure of Example 1 was followed except that for allele-specific HLA-B27 group amplification the same first 5' primer was used (SEQ.ID:1) with the 3' primer 5'-CGGTCAGTCTGTGCCTT-3'(SEQ.ID:3), . (see Figure 4) with 1 mM magnesium chloride in the amplification buffer, anealing at 52°C, and the product detected by ethidiu bromide staining following agarose gel electrophoresis and visualisation of a band of 210 base pairs in size.
  • Example 1 The procedure of Example 1 was conducted except using the primers 5'-CCGGAACACACAGATCTT-3' (SEQ.ID: ) and 5'-GTCGTAGGCGGACTGGTC-3' (SEQ.ID:5) (see Figure 3) for allele-group-specific amplification of HLA-Bw55 and HLA-B35 alleles.
  • the same procedure of Example 1 was conducted except that the magnesium chloride was provided at a concentration of 1 mM and 35 cycles were performed of 90 seconds at 94°C, 120 seconds at 58°C (to anneal) and 120 seconds at 72°C.
  • Prehybridisation using 1.8 x SSC, standardised sodium citrate, 10 x Denhardts solution, 2 mg/ml salmon sperm DNA and 0.2% SDS
  • hybridisation using 1.8 x SSC with 0.1% SDS
  • the probes used were:
  • Position 1 represents a cloned negative control (HLA-Bw53)
  • position 2 a cloned HLA-B27 allele.
  • Samples in positions 9, 14, 15 and 24 were amplified from individuals who did not have HLA-B27 on serological typing. All other samples were taken from HLA-B27 carriers.
  • HLA sub-type HLA-B2703 has been reported in a single American black. To look for this sequence in the Gambia an oligonucleotide which spans amino acid 59 of the alpha 1 domain was synthesised, which is the only position at which HLA-B*2703 differs from the most common Caucasian HLA-B27 allele, HLA-B*2705 ( Figure 4) . Surprisingly, it was found that 11 of the 18 HLA-B27 positive samples from- the Gambia hybridised to this oligonucleotide. All non-HLA-B27 controls did not.
  • HLA class I alleles have unique sequences which will allow their detection by a single oligonucleotide probe. Many are composed of patchwork type sequences where a combination of sequence motifs defines the allelic type.
  • HLA-Bw53 differs from another common allele, HLA B35, only by the presence of a short sequence at the 3' end of exon 2 that is shared by many other alleles, and which specifies the serological Bw4 epitope.
  • all of these alleles can also be typed using PCR by employing specific amplification primers.
  • HLA-B27 associated diseases in sub- Saharan Africa has been thought to be related to the near absence of this HLA type in unmixed Africans. This is not the case in the Gambia where nearly 3% of the population carry HLA-B27.
  • An alternative and attractive explanation raised by this study is that the African sub-type of HLA-B27 , B*2703 , carries a lesser risk of ankylosing spondylitis and other HLA-B27 related conditions.
  • Previously HLA-B*2703 has only been identified previously in one healthy American black.
  • Four of the other 5 sub-types of HLA-B27 have been shown to be as s oc iated with ankyl os ing spondylitis.
  • T lymphocytes that respond to the common sub-types HLA- B*2703 and HLA-B*2702 in both alloreactive and peptide specific ⁇ ytotoxic T cell ( CTL) assays fail to recognise HLA-B*2703. This would be predicted by the model of HLA-B27 disease pathogenesis which implicates cytotoxic lymphocytes reactive with an HLA-B27 specific peptide carried by foreign pathogens that crossreact with a similar or identical peptide normally expressed in joint tissues , the "arthritogenic peptide" model of ankylosing spondyl itis and reactive arthritis .
  • HLA-B*2703 The importance of determining whether or not HLA-B*2703 is associated with a high risk of ankylos ing spondyl itis arises from the s ingle amino acid difference between this allele and the common Caucasian sub-type HLA-B*2705, a substitution of histidine for tyrosine at position 59 of the alpha 1 domain ( Figure 4) . If the former is not a susceptibility allele this would strongly* support an arthritogenic peptide model of disease pathogenesis rather than the "altered-self" and "molecular mimicry" models which propose a crucial role for the cysteine residue at position 71 or neigbouring residues. Furthermore, it would provide a potential method of screening peptides isolated from microorganisms which represent HLA-B27 epitopes for possible involvement in disease pathogenesis.
  • HLA-B27 typing and sub-typing of numerous blacks with HLA-B27 related diseases may define the risk of disease associated with HLA-B*2703.
  • the availability of a simple method of HLA class I typing and sub-typing may facilitate such studies.
  • SEQ.ID NO. SEQUENCE TYPE: * nucleotide with corresponding protein
  • SEQ.ID NO. SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 21 base pairs STRANDEDNESS: single TOPOLOGY: linear PROPERTIES: primer complementary to nucleic acid corresponding to amino acids
  • SEQ.ID NO. SEQUENCE TYPE: nucleotide with corresponding protein
  • SEQ.ID NO. SEQUENCE TYPE: protein SEQUENCE LENGTH: 6 amino acids PROPERTIES: residues 2-8 in alpha l domain of
  • SEQ.ID NO. SEQUENCE TYPE: protein SEQUENCE LENGTH: 6 amino acids PROPERTIES: residues 85-90 in alpha 1 domain of
  • SEQ.ID NO.: 8 SEQUENCE TYPE: protein SEQUENCE LENGTH: 6 ⁇ ⁇ amino acids
  • PROPERTIES residues 67-72 in alpha 1 domain of
  • SEQ.ID NO. SEQUENCE TYPE: protein SEQUENCE LENGTH: 7 amino acids PROPERTIES: residues 61-67 in alpha l domain of
  • SEQUENCE TYPE protein SEQUENCE LENGTH: 6 amino acids
  • PROPERTIES residues 114-120 in alpha 2 domain of HLA-BW53 and HLA-B35
  • SEQ.ID NO. 11 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 18 base pairs STRANDEDNESS: single TOPOLOGY: linear PROPERTIES: probe specific for HLA-B27 group
  • SEQ.ID NO. 12 SEQUENCE TYPE: nucleotide with corresponding protein
  • SEQ.ID NO. 13 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 18 base pairs STRANDEDNESS: single TOPOLOGY: linear PROPERTIES: probe specific for region corresponding to Bw4 epitope

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Abstract

On décrit une technique d'amplification au moyen d'une réaction en chaîne de la polymérase (PCR) servant à amplifier les allèles HLA Classe I, uniquement un groupe d'allèles de HLA Classe I (par exemple le HLA-B27), ou bien un seul allèle spécifique, par exemple le HLA-B*2703. Le procédé utilise un tampon renfermant environ 10 % de DMSO ainsi qu'un chélateur, et peut amplifier l'ADN génomique. On peut utiliser des sondes oligonucléotidiques présentant la même spécificité que les amorces pour détecter des allèles ou des groupes d'allèles afin de déterminer si le risque est important ou peu important qu'une personne souffre d'arthrite.
EP91918606A 1990-11-05 1991-11-05 Procede d'amplification d'acide nucleique de hla classe i Expired - Lifetime EP0556214B1 (fr)

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EP98108382A EP0875582B1 (fr) 1990-11-05 1991-11-05 Procédé pour le typage de l'ADN de l'HLA de classe 1 par amplfication PCR

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GB9024005 1990-11-05
GB909024005A GB9024005D0 (en) 1990-11-05 1990-11-05 Process for amplifying nucleic acid
PCT/GB1991/001935 WO1992007956A1 (fr) 1990-11-05 1991-11-05 Procede d'amplification de l'acide nucleique

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EP0540997A1 (fr) * 1991-11-05 1993-05-12 F. Hoffmann-La Roche Ag Méthodes et réactifs pour le typage de l'ADN de l'HLA de classe I
ATE169682T1 (de) * 1993-03-18 1998-08-15 Innogenetics Nv Verfahren zur hla-b typisierung mittels spezifischer primer und probensätzen
US6413747B1 (en) * 1994-10-03 2002-07-02 Shimadzu Corporation Enhancement of nucleic acid amplification by the addition of a polyamine
US5550039A (en) * 1995-03-07 1996-08-27 Hoffmann-La Roche Inc. Oligonucleotide primers for HLA class I B locus DNA typing
US5753442A (en) * 1995-09-01 1998-05-19 Cedars-Sinai Medical Center Method for determining genetic predisposition for seronegative spondyloarthropathies and products useful therefor
GB9524381D0 (en) * 1995-11-29 1996-01-31 Anthony Nolan Bone Marrow Trus Method for identifying an unknown allele
WO1997031126A1 (fr) * 1996-02-20 1997-08-28 The Perkin-Elmer Corporation Procede et reactifs de typage de genes hla de classe i
US6828094B2 (en) * 1996-12-20 2004-12-07 Roche Diagnostics Gmbh Method for the uncoupled, direct, exponential amplification and sequencing of DNA molecules with the addition of a second thermostable DNA polymerase and its application
DE19715430A1 (de) * 1997-04-14 1998-11-26 Boehringer Mannheim Gmbh Verfahren zur Typisierung von Allelen
FR2793809B1 (fr) 1999-05-20 2006-07-28 Biomerieux Sa Procede d'analyse de la predisposition genetique d'un patient a au moins une maladie et amplification adaptee a un tel procede
US6783940B2 (en) * 2001-10-31 2004-08-31 Applera Corporation Method of reducing non-specific amplification in PCR
JP4485367B2 (ja) 2002-12-03 2010-06-23 パソゲン リムーバル アンド ダイアグノスティック テクノロジーズ インコーポレイテッド プリオンタンパク質リガンドおよび使用方法
WO2009108626A2 (fr) 2008-02-25 2009-09-03 Eyegate Pharma S.A.S. Apport amélioré d'une substance thérapeutique à des tissus oculaires par iontophorèse
EP2285982B1 (fr) * 2008-05-27 2018-04-11 Dako Denmark A/S Compositions et procédés pour la détection d' aberrations chromosomiques avec de nouveaux tampons d' hybridation
WO2010097655A1 (fr) 2009-02-26 2010-09-02 Dako Denmark A/S Compositions et procédés pour des applications d'hybridation d'arn
ES2601237T3 (es) * 2009-12-02 2017-02-14 Dako Denmark A/S Composiciones y métodos para realizar hibridaciones sin desnaturalización
US10662465B2 (en) 2011-09-30 2020-05-26 Agilent Technologies, Inc. Hybridization compositions and methods using formamide
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EP0875582B1 (fr) 2007-06-27
DE69131101D1 (de) 1999-05-12
GB9024005D0 (en) 1990-12-19
DE69131101T2 (de) 1999-08-12
US5824515A (en) 1998-10-20
EP0875582A2 (fr) 1998-11-04
US5525492A (en) 1996-06-11
EP0875582A3 (fr) 2004-03-24
DE69133573D1 (de) 2007-08-09
EP0556214B1 (fr) 1999-04-07

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